PROJECT SUMMARY The overarching goal of this five-year Career Development Award proposal is to support Dr. Jillian Ziemanski's development into an independent ocular surface immunologist with expertise in dry eye disease, particularly Sjgren's syndrome. Primary Sjgren's syndrome (pSS) is a chronic autoimmune exocrinopathy and epithelitis that leads to drying of the ocular and oral mucosae. Patients often present with severe dry eye disease that may be associated with decreased vision, corneal ulceration, and corneal scarring. The complement cascade, though best known for its role in infection, has been shown to be aberrantly and strongly activated in several autoimmune diseases. Activation of complement can lead to in situ killing of cells by disruption of their plasma membranes, removal of viable cells by phagocytosis, and inflammation, a process that, in turn, further upregulates complement protein production. Inflammation and conjunctival cell death are hallmarks of pSS dry eye disease (pSS-DE). Further, pSS-DE is known to have a 9:1 predilection for females. The central hypothesis of this project is that the complement system is primed for activation in all types of inflammatory dry eye disease and may be further potentiated by estradiol levels. The system, however, likely remains inactive in non-pSS-DE but is activated in pSS-DE by immune complexes. This activation may serve as a potential biomarker for differentiation between pSS- and non-pSS-DE and as a mechanism to target for future therapeutic intervention. Therefore, the Specific Aims for this project are (Aim 1) to evaluate complement protein production by immortalized human conjunctival epithelial cells stimulated with dry eye-associated cytokines, (Aim 2) to assess differences in total inducible complement activity of the tears in vitro with respect to serum estradiol levels, and (Aim 3) to assess differences in complement activation at the ocular surface in pSS-DE versus non-pSS-DE. The training activities and research project will take place in the interdisciplinary, collaborative environment of the University of Alabama at Birmingham. The multidisciplinary mentoring and consulting team is comprised of a dry eye disease, tear film proteomics, and ocular surface expert (Dr. Kelly Nichols, primary mentor); an expert complement biologist and immunologist (Dr. Alex Szalai, co-mentor); an expert immunologist experienced with flow cytometric analysis (Dr. Chander Raman, consultant); and an expert in complement-based diagnostic device development (Dr. Scott Barnum, consultant). Upon completion of this five-year K23 project, Dr. Ziemanski will have had one- on-one training in complement biology, immunology, tear film proteomics, flow cytometric analysis, and diagnostic device development. This K23 project will support an R01 submission to fully investigate the mechanisms of complement activation and regulation on the ocular surface in pSS-DE and will serve as an impetus to her career as an independent ocular surface immunologist poised to investigate mechanisms of, assess treatments for, and develop innovative diagnostic devices for pSS-DE.